Leydig cells in the testis are the primary source of testosterone in the male. Our previous studies established three intermediate stages of the Leydig cell lineage in rats: a mesenchymal-like, highly proliferative progenitor Leydig cell (PLC), first observed at day 10-11 postpartum that is recognized as steroidogenic through histochemical staining of the marker enzyme 3(3-hydroxysteroid dehydrogenase (3|3- HSD); an immature Leydig cell (ILC) that has mitochondrial and smooth endoplasmic reticulum membrane structures typical of steroid secreting cells but that synthesizes primarily Sa-reduced androgens rather than testosterone as end products; and an adult Leydig cell (ALC) that is proliferatively quiescent and terminally differentiated for testosterone secretion. A fundamental unanswered question is whether the ultimate source of adult Leydig cells is a small pool of undifferentiated, self-renewing stem Leydig cells (SLCs). The experiments in this application build on our recent identification and isolation of putative SLCs from postpartum day 7 rat testes. These cells do not express the Leydig cell markers LH receptor (LHR) or 3(3- HSD, do not synthesize steroids, are capable of at least 60 self-renewing divisions in vitro when cultured in the presence of leukemia inhibitory factor (LIF), and upon appropriate stimulus, form PLCs that synthesize testosterone. Most importantly, when transplanted into Leydig cell-depleted adult rat testes, these putative stem cells repopulate the interstitium and later express 3(3-HSD. Building on these initial observations, we propose herein: 1) to identify the mechanisms that lead the neonatal SLCs to undergo renewal, commit to the steroidogenic lineage, and undergo terminal differentiation; 2) identify the cellular and molecular changes involved in the acquisition of steroidogenic capacity when Leydig cells differentiate from SLCs; and 3) examine the ability and mechanisms by which SLCs self-renew and differentiate in vivo when implanted into the testes of host rats. Throughout these aims we will ask whether SLCs isolated from adult animals retain the same properties of sternness observed in the neonatal SLCs. When completed, this project will provide both a line of defined cells that can be used by the research community to further understand the development of steroidogenic cells, and perhaps a new therapeutic tool for androgen replacement in hypogonadal and aging males. [unreadable] [unreadable] [unreadable]